Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

Abstract

SH2 (src‐homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein‐effector coupling is provided by the association of phosphatidylinositol 3‐kinase (PI 3‐kinase) with specific phosphorylation sites within the PDGF receptor, the c‐Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS‐1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3‐kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor‐, IRS‐1‐ and c‐Src‐derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3‐kinase. Although phosphotyrosine and both activating and non‐activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non‐local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.

CD38 Signaling in T Cells Is Initiated within a Subset of Membrane Rafts Containing Lck and the CD3-ζ Subunit of the T Cell Antigen Receptor

“…Because CD3-and CD3-⑀ are tyrosine-phosphorylated upon CD38 cross-linking (Zubiaur et al (28,31) and this paper), and these proteins could interact in a phosphorylationdependent manner with the p85␣ PI 3-kinase (31,79,80), they could target PI 3-kinase to rafts. In addition, binding of PI 3-kinase to the TCR-CD3 per se could up-regulate the PI 3-kinase activity (79,80), presumably by a conformational change as reported previously for other p85-binding proteins (81,82). Other candidate molecules are LAT, Shc, and Cbl, as these molecules bind p85 (83), and are tyrosine-phosphorylated upon CD38 stimulation (Figs.…”

New insights into protein‐tyrosine kinase receptor signaling complexes

“…Presumably, following binding of the phosphopeptide to one or both of the p85 SH2 domains, a conformational change takes place and is transmitted to the associated pl10 catalytic subunit, resulting in an increase in PI 3-kinase activity. Small conformational changes in p85, and an isolated N-terminal SH2 domain from this protein, have been observed upon phosphopeptide binding using circular dichroism and fluorescence studies Shoelson et al, 1993).…”

Kinetic Analysis of Platelet-derived Growth Factor Receptor/Phosphoinositide 3-Kinase/Akt Signaling in Fibroblasts

“…The 110 kDa ␣ and ␤ isoforms of the PI 3-kinase catalytic subunit are involved in receptor-tyrosine kinase signaling, and they almost exclusively phosphorylate PtdIns(4,5)P 2 to form PtdIns(3,4,5)P 3 , with PtdIns(3,4)P 2 generated from the subsequent dephosphorylation of PtdIns(3,4,5)P 3 . The 85 kDa regulatory subunit coordinates binding of PI 3-kinase to PDGF receptors through dual SH2 domains, transmitting a conformational change that activates the catalytic subunit (17)(18)(19)(20). Equally if not more important is the induced localization of PI 3-kinase in proximity to its plasma membrane-associated substrate (21).…”